published_or_final_version / Medicine / Master / Master of Research in Medicine

Using tricaprylin (TC) and trilinolenin (TLN) as substrates, biosynthesis of medium-long-medium (MLM) type structured lipids (SLs), by Lipozyme IM from Rhizomucor meihei and Novozym 435 from Candida antarctica , was investigated to determine their capacity as biocatalysts for the biosynthesis of SLs. At 30°C, Lipozyme IM showed higher bioconversion yield (24.7%) and initial enzyme activity (6.3 mumol CLnC/g enzyme/min) as compared to that of 24.0% and 1.6 mumol CLnC/g enzyme/min, respectively, for the Novozym 435 at 50°C. As a result, Lipozyme IM was subsequently used for further investigations. The SLs were recovered and characterized by silver-ion exchange high-performance liquid chromatography and gas-liquid chromatography. The structural analyses indicated that the major products of the enzymatic reaction were 1,3-dicapryl-2-linolenyl glycerol (CLnC) and 1(3)-capryl-2,3(1)-dilinolenyl glycerol (CLnLn). In order to optimize the bioconversion yield of CLnC, selected parameters, including initial water activity and solvent type, lipase concentration (5 to 20 mg solid enzyme), substrate molar ratios (TC:TLN of 1:4 to 8:1) and molecular sieve (5 to 20 mg/mL, Type 3A), were investigated. The experimental results showed that using hexane at initial aw 0.06, 10 mg solid enzyme/mL and substrate molar ratio of TC to TLN of 6:1 resulted in the highest bioconversion yield of 73.2% of CLnC. However, the addition of molecular sieve to the reaction medium resulted in a 14.0% decrease in the bioconversion yield of CLnC. Using the optimized conditions, the effects of TLN concentration and other selective limiting parameters, including the denaturation of enzyme, aw and the formation of glycerol layer, on the mass productivity (PM), enzymatic productivity (PE) and volumetric productivity (PV) of the interesterification reaction were investigated. Using 80 mM TLN, the maximum PM of 15.5 mg CLnC/g substrates/h was obtained; however, using 200 mM TLN, the maximum PE and PV were 0.07 mg/enzyme unit/h and 6.1 g CLnC/L/h, respectively. The addition of 3 mg Silica gel to the reaction medium resulted in 52.0, 37.3 and 37.3% increase in PM, PE and PV, respectively.

No description available.

Lipid accumulation is a pathological feature of every type of kidney injury. However, despite this striking histological feature, physiological accumulation of lipids in the kidney is poorly understood. We studied whether the accumulation of lipids in the fasted kidney is derived from lipoproteins or non-esterified fatty acids (NEFAs). Increasing circulating NEFAs using a beta adrenergic receptor agonist caused a 15-fold greater accumulation of lipid in the kidney, while mice with reduced NEFAs due to adipose tissue deficiency of adipose triglyceride lipase had reduced renal triglycerides. Fasting-induced kidney lipid accumulation was not affected by inhibition of lipoprotein lipase (LpL) with poloxamer 407 or by use of mice with induced genetic LpL deletion. Despite the increase in CD36 expression with fasting, genetic loss of CD36 did not alter fatty acid uptake or triglyceride accumulation. Our data demonstrate that fasting-induced triglyceride accumulation in the kidney correlates with the plasma concentrations of NEFAs but is not due to uptake of lipoprotein lipids and does not involve the fatty acid transporter CD36. A second project was initiated to assess how diabetes causes increased systemic inflammation. Calgranulins S100A8 and S100A9 circulating levels are increased during diabetes and might instigate a sterile inflammatory response in the innate immune system. To determine whether krüppel-like factor 5 (KLF5) regulates S100A8 and S100A9 during hyperglycemia; we generated myeloid-specific KLF5 knockout mice (MKK) and found these mice had no change in circulating monocytes and neutrophils. We isolated neutrophils from these mice and found that S100A8 and S100A9 expression was not changed. We found similar null results when these mice were made diabetic. We conclude that this line of myeloid-deficient KLF5 knockout mice do not have changes in S100A8 or S100A9 expression or in the numbers of circulating white cells.

The absorption, packaging, and delivery of fat to appropriate peripheral tissues is essential for maintaining metabolic homeostasis, and defects or dysregulation of these processes can contribute to metabolic disorders such as diabetes, obesity, and hyperlipidemia. In the intestine, dietary fat is packaged into triglyceride-rich lipoprotein particles and delivered to peripheral tissues through the circulatory system. Lipolysis of lipoprotein triglycerides requires the enzyme lipoprotein lipase (LPL) and takes place on the luminal surface of capillary endothelial cells. Lipolysis by LPL is regulated in part by two proteins, GPIHBP1 and ANGPTL4. GPIHBP1, a GPI-anchored protein of capillary endothelial cells, is responsible for transporting LPL across endothelial cells to the capillary lumen. Without this transport, LPL becomes mislocalized to the interstitial space and cannot access triglyceride-rich lipoproteins, resulting in severe hypertriglyceridemia. Conversely, ANGPTL4 inhibits LPL and ANGPTL4 deficiency results in increased LPL activity and lower plasma triglyceride levels. Our goal is to understand how the interactions between LPL, GPIHBP1, and ANGPTL4 influence the delivery of triglyceride-derived fatty acids to tissues. In this thesis, I (1) use mouse models to elucidate the function of ANGPTL4 in regulating the clearance of diet-derived fat from plasma, (2) describe a mechanism for GPIHBP1-independent plasma triglyceride clearance observed in mice lacking both GPIHBP1 and ANGPTL4, and (3) propose that this GPIHBP1-independent mechanism is also operative in Gpihbp1–/– mice following a high fat diet challenge. The contributions of this thesis are significant because they close a gap in our knowledge of how and where ANGPTL4 functions, as well as indicating that, when ANGPTL4 is suppressed or absent altogether, a GPIHBP1-independent mechanism can function to clear plasma triglycerides.

Oral consumption of flaxseed improves serum lipid parameters, and the flaxseed lignan, secoisolariciresinol diglucoside (SDG) may mediate these effects. SDGs therapeutic potential cannot be fully realized until its mechanism of action and pharmacokinetics are more completely characterized.</p>This research aimed to assess SDGs effects in dietary models of hypertriglyceridemia, hypercholesterolemia, and obesity. Furthermore, this thesis work provided preliminary pharmacokinetic data on SDGs aglycone form, secoisolariciresinol (SECO). Dietary manipulations were used to induce hyperlipidemic states in female Wistar or male Sprague-Dawley rats. Groups of 10 rats were randomly assigned to one of three (obesity and cholesterol diets) or four (fructose) treatment groups: 1) Normal diet with 0.0 µmol SDG/kg body weight; 2) Dietary manipulation with 0.0 µmol SDG/kg; 3) Dietary manipulation with 4.4 µmol SDG/kg; and 4) 10% fructose in water with 8.8 µmol SDG/kg. Lignan or vehicle (saline) was administered daily by oral gavage for four weeks (2 weeks for male Sprague-Dawleys). After four (or two) weeks of SDG administration, body and liver weights were recorded, serum lipids were measured using enzymatic kits, hepatic fat accumulation was determined by histochemical analysis and hepatic mRNA expression of triglyceride pathway targets was evaluated using real-time RT-PCR. A 10% fructose in water model was effective for the induction of hypertriglyceridemia in male Sprague-Dawley rats but ineffective in female Wistar rats of similar age. Neither 4.4 nor 8.8 µmol SDG/kg improved serum and hepatic triglyceride parameters in male Sprague-Dawley rats on a 10% fructose in water diet. It is suspected that gender and strain are important factors for this model of hypertriglyceridemia. Dietary manipulations for the induction of hypercholesterolemia and obesity in female Wistar rats were not effective following 4 weeks administration of a 1% cholesterol diet and a 45% fat diet respectively. Since previous studies were able to successfully induce hypercholesterolemia in the same model, it is suspected that the differences in age of the animals accounted for the inconsistent results. Strain, gender and age of animals were identified as important considerations when trying to induce hyperlipidemic states through dietary manipulations.</p>SDG (4.4 µmol/kg) dosed daily for four weeks caused no gross morphological organ changes or alterations in blood chemistry or hematology parameters. Following an intravenous bolus (10 mg/kg), secoisolariciresinol (SECO) disposition was consistent with two-compartment pharmacokinetics, with distribution and elimination half-lives at 26 seconds and 5 minutes, respectively. No SECO was detected in the plasma following an oral bolus (10 mg/kg). Further investigation into SDGs hypolipidemic effects are required to elucidate its mechanism of action. A complete pharmacokinetic study is warranted to fully understand SDGs safety and efficacy.

Oral consumption of flaxseed improves serum lipid parameters, and the flaxseed lignan, secoisolariciresinol diglucoside (SDG) may mediate these effects. SDGs therapeutic potential cannot be fully realized until its mechanism of action and pharmacokinetics are more completely characterized.</p>This research aimed to assess SDGs effects in dietary models of hypertriglyceridemia, hypercholesterolemia, and obesity. Furthermore, this thesis work provided preliminary pharmacokinetic data on SDGs aglycone form, secoisolariciresinol (SECO). Dietary manipulations were used to induce hyperlipidemic states in female Wistar or male Sprague-Dawley rats. Groups of 10 rats were randomly assigned to one of three (obesity and cholesterol diets) or four (fructose) treatment groups: 1) Normal diet with 0.0 µmol SDG/kg body weight; 2) Dietary manipulation with 0.0 µmol SDG/kg; 3) Dietary manipulation with 4.4 µmol SDG/kg; and 4) 10% fructose in water with 8.8 µmol SDG/kg. Lignan or vehicle (saline) was administered daily by oral gavage for four weeks (2 weeks for male Sprague-Dawleys). After four (or two) weeks of SDG administration, body and liver weights were recorded, serum lipids were measured using enzymatic kits, hepatic fat accumulation was determined by histochemical analysis and hepatic mRNA expression of triglyceride pathway targets was evaluated using real-time RT-PCR. A 10% fructose in water model was effective for the induction of hypertriglyceridemia in male Sprague-Dawley rats but ineffective in female Wistar rats of similar age. Neither 4.4 nor 8.8 µmol SDG/kg improved serum and hepatic triglyceride parameters in male Sprague-Dawley rats on a 10% fructose in water diet. It is suspected that gender and strain are important factors for this model of hypertriglyceridemia. Dietary manipulations for the induction of hypercholesterolemia and obesity in female Wistar rats were not effective following 4 weeks administration of a 1% cholesterol diet and a 45% fat diet respectively. Since previous studies were able to successfully induce hypercholesterolemia in the same model, it is suspected that the differences in age of the animals accounted for the inconsistent results. Strain, gender and age of animals were identified as important considerations when trying to induce hyperlipidemic states through dietary manipulations.</p>SDG (4.4 µmol/kg) dosed daily for four weeks caused no gross morphological organ changes or alterations in blood chemistry or hematology parameters. Following an intravenous bolus (10 mg/kg), secoisolariciresinol (SECO) disposition was consistent with two-compartment pharmacokinetics, with distribution and elimination half-lives at 26 seconds and 5 minutes, respectively. No SECO was detected in the plasma following an oral bolus (10 mg/kg). Further investigation into SDGs hypolipidemic effects are required to elucidate its mechanism of action. A complete pharmacokinetic study is warranted to fully understand SDGs safety and efficacy.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2004. / Vita. Includes bibliographical references (leaves 181-227).

The triglyceride-free fatty acid (TG-FFA) cycle was studied in white adipose tissue. The major aims of the study were 1) to see if the rate of TG-FFA cycling (i.e. FFA reesterification) and the sensitivity properties (see Newsholme and Crabtree, 1976, Biochem. Soc. Symp. 41, 61-109) were affected by various treatments, and 2) to measure the rate of cycling in vivo and assess its contribution to the metabolic rate of an animal. There are two ways of estimating the rate of TG-FFA cycling; the first is based on the release of glycerol and FFA from the tissue, and the second on the synthesis of the glycerol and FFA moieties of triglyceride. Experimental agreement between the two methods is very good. It is shown that the rate of TG glycerol synthesis can be estimated by measuring the incorporation of tritium from tritiated water into the TG-glycerol moiety; this method is used to study the TG-FFA cycle in vivo. Experimental results indicated that the rate of TG-FFA cycling in white adipose tissue in vitro and in vivo is affected by various short- and long-term treatments. However, the reesterification of FFA in adipose tissue can only account for perhaps ~1% of the basal metabolic rate of a mouse, and perhaps 4% of the increase in osygen consumption observed in fenoterol-treated mice. The equations of Newsholme and Crabtree (1976) describing the sensitivity properties of substrate cycles are extended and used to show that the TG-FFA cycle increases the sensitivity of control of FFA release from adipose tissue. The degree of sensitivity attainable is variable depending on the treatment used. The use of tritiated water for estimating TG-FFA cycling is tentatively extended to brown adipose tissue. It is suggested that the rate of cycling could be used as an indicator of sympathetic activity in brown and white adipose tissue.

Thesis (Ph.D.)-- University of Wisconsin--Madison, 2007. / Includes bibliographical references. Also available on the Internet.